Stationary phases and a purification process using the stationary phases

ABSTRACT

This invention relates to a novel stationary phase of Formula I and a method for purifying a peptide or lipopeptide in liquid chromatography using select stationary phases, including the stationary phases of Formula I to improve the resolution and/or productivity of the purification. This chromatographic method can be used for either an analytical or preparative scale purification.

BACKGROUND OF THE INVENTION

Lipopeptides, such as Pneumocandin B₀, are often the product of afermentation process. During such a fermentation process, many closelyrelated analogues are produced along with the desired product. Liquidchromatography systems are frequently used to purify the crudefermentation product. A liquid chromatography system usually consists ofa stationary phase and a mobile phase. For purification of a peptide orlipopeptide, the stationary phase can be silica gel, alumina or othermaterials, and the mobile phase can be a single solvent or a mixture ofsolvents, which includes organic solvents and water.

Silica gel chromatography and other types of liquid chromatography areuseful for separating these analogues. However, in practice, theseparation of certain closely related analogues from the desired productis often un-satisfactory, because of poor chromatographic resolution,i.e. overlap of chromatographic peaks. To achieve the desired purity ofthe main product at a reasonable yield requires restricting the amountof material (often referred to as feed or column load) loaded onto thecolumn per run, which limits the productivity of the operation.

The chromatographic purification of Pneumocandin B₀ has historicallybeen difficult owing to poor chromatographic resolution. Thechromatography utilizes a mobile phase consisting of a mixture ofsolvents, specifically ethyl acetate (EtOAc), methanol (MeOH) and water,on a silica gel column. In the past, separation of key impurities, suchas that of Pneumocandins B₅ and E₀ from Pneumocandin B₀, was difficultowing to poor chromatographic resolution. Some analogs were onlypartially resolved from the main product peak using preparativeconditions. To achieve the desired product purity with this limitedresolution required that the purification step be run with low columnloading, which limited productivity.

Pneumocandin B₀, with a molecular weight of 1065 Daltons, is a naturalproduct and serves as an intermediate in the production of Caspofunginacetate (Cancidas®). Pneumocandin B₀ is produced as a secondarymetabolite by fermentation of the fungus Glarea lozoyensis. See U.S.Pat. Nos. 5,194,377 and 5,202,309. The structures of Pneumocandin B₀ andthree of the key analog impurities, all comprised of a cyclichexapeptide coupled with dimethylmyristate side chain, are shown inTable 1. TABLE 1 Pneumocandin B₀ and three of its analogs

Compound R¹ R² R³ R⁴ R⁵ R⁶ Pneumocandin B₀ OH OH Me OH H OH PneumocandinB₅ OH H Me OH H OH Pneumocandin C₀ OH OH Me OH OH H Pneumocandin E₀ OHOH Me OH H H

Silica gel chromatography exploits the subtle variations in bindingaffinity of the hydroxy-rich cyclic hexapeptide core of the desiredproduct and the analog impurities, including Pneumocandins B₅, C₀, andE₀, to effect a separation. In the silica gel HPLC purification,Pneumocandins B₅ and E₀, two of the key analog impurities co-produced inthe fermentation of Pneumocandin B₀, elute very closely to PneumocandinB₀. Therefore, to meet the target impurity levels in the purifiedmaterial for these and similar analogs, the quantity of crudePneumocandin B₀ that can be loaded onto the column is limited. As aresult, significant efforts have been made to improve the resolution ofkey impurities. For instance, the ternary ethyl acetate-methanol-watermobile phase has been balanced to optimize resolution betweenPneumocandin B₀ and key analog impurities. D. J. Roush, F. D. Antia, K.E. Göklen J. Chromatography A, 827 (1998) 373-389. Additionally, the useof a mobile phase modifier has been demonstrated to enhance theresolution and selectivity between Pneumocandin B₀ and key analogimpurities. See J. Nti-Gyabaah, et al., “Large-scale purification ofpneumocandin B₀, a precursor for CANCIDAS”, PREP-2003, 16thInternational Symposium, Exhibit and Workshops on Preparative/ProcessChromatography, San Francisco, Calif., Wednesday, Jul. 2, 2003 or U.S.Provisional Application No. 60/422,356 filed Oct. 30, 2002.

SUMMARY OF THE INVENTION

This invention relates to a novel stationary phase of Formula I and amethod for the purification of a peptide or a lipopeptide by using aliquid chromatography system with select stationary phases, includingthe stationary phases of Formula I and a mobile phase, to improve theselectivity and/or productivity of the purification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1:

Chromatogram (absorbance 278 nm vs. time) of silica gel chromatographicpurification of crude Pneumocandin B₀.

FIG. 2:

Chromatogram (absorbance 278 nm vs. time) of silica gel chromatographicpurification of crude Pneumocandin B₀ with a proline-modified mobilephase.

FIG. 3:

Chromatogram (absorbance 278 nm v. time) of aminopropyl-silica gelchromatographic purification of crude Pneumocandin B₀.

FIG. 4:

Chromatogram (absorbance 278 nm vs. time) of Amide-80-silica gelchromatographic purification of crude Pneumocandin B₀.

FIG. 5:

Capacity factors of Pneumocandin B₀ and related analogs on silica gel,silica gel-proline modified, aminopropyl silica gel and Amide-80-silicagel.

FIG. 6:

Chromatogram (absorbance 278 nm vs. column volumes) for a silica gelchromatography of crude Pneumocandin B₀ using a proline-modified mobilephase 88:9:7 ethyl acetate:methanol:water eluent with 0.12 g/L proline(presaturated column) and 75:17:8 ethyl acetate:methanol:water as feedsolvent mixture with 1.5 g/L proline in the feed.

FIG. 7:

Chromatogram (absorbance 278 nm vs. column volumes) for anAmide-80-silica gel chromatography of crude Pneumocandin B₀ using phase88:9:7 ethyl acetate:methanol:water eluent and 75:20:8 ethylacetate:methanol:water as feed solvent mixture.

FIG. 8:

Chromatogram (absorbance 278 nm vs. column volumes) forN-L-prolyl-3-aminopropyl silica gel chromatography of crude PneumocandinB₀ using phase 88:9:7 ethyl acetate:methanol:water eluent and 75:20:8ethyl acetate:methanol:water as feed solvent mixture.

FIG. 9:

Chromatogram (absorbance 278 nm vs. column volumes) forN-methylcarbamoyl-3-aminopropyl silica gel chromatography of crudePneumocandin B₀ using 88:9:7 ethyl acetate:methanol:water eluent and75:20:8 ethyl acetate:methanol:water as feed solvent mixture.

FIG. 10:

Chromatogram (absorbance 278 nm vs. column volumes) forN-β-alaninamidopropyl silica gel chromatography of crude Pneumocandin B₀using 88:9:7 ethyl acetate:methanol:water eluent and 75:20:8 ethylacetate:methanol:water as feed solvent mixture.

DETAILED DESCRIPTION OF THE INVENTION

A stationary phase of Formula I:

wherein

-   R is:

a) —(CH₂)_(n)CONH₂, or

b) —COOR¹;

-   n is: 1 to 4; and-   R¹ is: C₁-C₂ alkyl.

An embodiment of the stationary phases of Formula I are:

Additional stationary phases that have been useful in the purificationare: Amide-80 (manufactured by Tosoh Biosep LLC., Japan),

manufactured by Eka Chemicals of Sweden

-   -   described in U.S. Pat. No. 6,342,160.

A method for the purification of a peptide or a lipopeptide by using aliquid chromatography system with a stationary phase selected from thegroup consisting of: the stationary phases of Formula I, as recitedabove, Amide-80,

and a mobile phase, to improve the selectivity and/or productivity ofthe purification is disclosed.

Additional stationary phases may be useful in the purification ofcertain peptides and lipopeptides, which fall within the scope offormula Ia as defined below:

wherein

-   R is:

a) H,

b) N-acetyl-D-Asparginyl,

c) D-Glutaninyl,

d) L-Prolinyl,

e) Iso-L-Glutaminyl,

f) —(CH₂)_(n)NH₂,

g) —(CH₂)_(n)CONH₂,

h) —CO(CH₂)_(n)CO₂H,

i) —CONH₂,

j) CONHR¹,

k) —COOR¹, or

l) —COR²;

-   n is: 1 to 4;-   R¹ is C₁-C₆ alkyl, unsubstituted or substituted with one, two or    three substituents selected from Cl, F, Br or I; and-   R² is C₁-C₆ alkyl, unsubstituted or substituted with one, two or    three substituents selected from Cl, F, Br or I, aryl, wherein aryl    is defined as phenyl or napthyl, unsubstituted or substituted with    one, two or three substituents selected from Cl, F, Br, I, or nitro.

Examples of lipopeptides, for which this purification process is useful,are echinocandin derivatives, such as Pneumocandin B₀, Caspofungin,Cilofungin and Micafungin as well as Anidulafungin and Daptomycin, andparticularly the natural product precursors cof Caspofungin, Micafungin,Cilofungin, Anidulafungin and Daptomycin. The naturalproduct/fermentation product precursor for Caspofungin is PneumocandinB₀. Caspofungin acetate (CANCIDAS®) is a semisynthetic lipopeptideechinocandin B derivative currently being sold in the US as anantifungal agent for intravenous administration. Anidulafungin is asemisynthetic lipopeptide echinocandin B derivative under development byEli Lilly/Versicor as an antifungal agent for intravenousadministration. Anidulafungin is disclosed in U.S. Pat. Nos. 5,965,525and 6,384,013, hereby incorporated by reference. Cilofungin is anechinocandin lipopeptide disclosed by Eli Lilly in U.S. Pat. No.4,293,489 for use as an antifungal agent, hereby incorporated byreference. Micafungin (FUNGARD™) is an echinocandin-like lipopeptideunder development by Fujisawa, as an antifungal agent for intravenousadministration. Micafungin is disclosed in U.S. Pat. No. 6,107,458hereby incorporated by reference. Daptomycin (CIDECIN™) is asemisynthetic lipopeptide derivative under development by Cubist as anantibacterial agent. Daptomycin is disclosed by Eli Lilly in U.S. Pat.No. 4,537,717 hereby incorporated by reference.

A liquid chromatography system employs a mobile phase and a stationaryphase. The mobile phase is a solvent system comprising one or moresolvents, the composition of which is either constant throughout thepurification process, or a gradient, where the solvent composition ischanged over time during the purification process. The mobile phasesolvents include, but are not limited to, water, methanol, ethanol,isopropanol, hexane, heptane, ethyl acetate, isopropyl acetate,acetonitrile, methyl t-butyl ether (MTBE) and methylene chloride. Thestationary phase is selected from the group consisting of: thestationary phases of Formula I:

wherein R is: —(CH₂)_(n)CONH₂, or —COOR¹; n is: 1 to 4; and R¹ is: C₁-C₂alkyl;Tosoh Amide 80,

The instant invention provides a chromatographic purification method fora peptide or lipopeptide, which employs a silica gel amino- oramide-containing stationary phase. A column volume (hereinafter referredto as cv) is defined as the volume of solvent needed to traverse thecolumn. Column load refers to the amount of material (crude lipopeptideor peptide) that is applied to the column is a single injection cycle.Column load may also be referred to as column feed or feed load.

The examples provided herein are intended to assist in a furtherunderstanding of the invention. Particular materials, employed speciesand conditions are intended to be further illustrative of the inventionand not limitative of the reasonable scope thereof.

EXAMPLE 1

Preparation of N-β-alaninamidopropyl silica

Kromasil amino silica (5 g, 10μ, 100 Å) was placed in a 100 mL roundbottom flask, to which was added 25 mL of dichloromethane. Followingcomplete wetting of the stationary phase, aided by gentle swirling, asolution of acrylamide containing 25-30 ppm cupric ion as a free radicalinhibitor (14 mMoles) in 25 mL of dichloromethane was added, and themixture was rotated overnight on a rotary evaporator apparatus at roomtemperature and without applied vacuum. The following morning, themixture was filtered on a sintered glass funnel, washed three times with30 mL of 20% methanol in dichloromethane, taken up in a slurry with2-propanol, and packed into a 4.6 mm id×25 cm length HPLC column forevaluation. Residual stationary phase taken from the column packerreservoir was dried overnight under high vacuum, then submitted forcombustion analysis (C 6.3%; N 1.8%).

EXAMPLE 2

In Situ Preparation of N-β-alaninamidopropyl silica

A commercial amino silica column (Chromegabond Amine; 5μ particle size;60 Å pore size; 4.6 mm column i.d.; 25 cm column length) was flushedwith dichloromethane at a flow rate of 2 mL/min for a period of 30minutes. Then a 10 M solution of acrylamide in acetonitrile wascirculated through the aminopropyl silica HPLC column at a flow rate of2 mL/ml n, the effluent from the column being directed to the pump inletreservoir so as to allow reagent recirculation. Flow of the reagentsolution was continued for a period of 4 hours, whereupon the column waswashed with a solution of first dichloromethane at 2 mL/min for 20minutes, then 20% methanol in dichloromethane at 2 mL/min for 20minutes.

EXAMPLE 3

Preparation of N-methylcarbamoyl-3aminopropyl silica

Kromasil amino silica (5 g, 10μ, 100 Å) was placed in a 100 mL roundbottom flask, to which was added 25 mL of dichloromethane. Followingcomplete wetting of the stationary phase, aided by gentle swirling, asolution of methyl chloroformate (10 mMoles) and triethylamine (10mMoles, 1.0 eq) in 25 mL of dichloromethane was then added, and themixture was rotated overnight on a rotary evaporator apparatus at roomtemperature and without applied vacuum. The following morning, themixture was filtered on a sintered glass funnel, washed three times with30 mL of 20% methanol in dichloromethane, taken up in a slurry with2-propanol, and packed into a 4.6 mm id×25 cm length HPLC column forevaluation. Residual stationary phase taken from the column packerreservoir was dried overnight under high vacuum, then submitted forcombustion analysis (C 6.0%; N 1.4%).

EXAMPLE 4

In Situ Preparation of N-methylcarbamoyl-3-aminopropyl silica

A commercial amino silica column (Chromegabond Amine; 5μ particle size;60 Å pore size; 4.6 mm column i.d.; 25 cm column length) was flushedwith dichloromethane at a flow rate of 2 mL/min for a period of 30minutes. A 1 M solution of the methyl chloroformate in dichloromethanecontaining one equivalent of triethylamine was then passed through thecolumn at a flow rate of 2 mL/min. The column was then flushed withdichloromethane for 10 minutes at 2 mL/min, followed by flushing with20% methanol in dichloromethane for 20 minutes at 2 mL/min.

EXAMPLE 5

Preparation of N-L-prolyl-3-aminopropyl silica

Kromasil amino silica (5 g, 10μ, 10 Å) was placed in a 100 mL roundbottom flask, to which was added 25 mL of dichloromethane. Followingcomplete wetting of stationary phase, aided by gentle swirling, asolution of Boc-L-Pro (4.7 mMoles) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 4.7mMoles, 1.0 eq) in 25 mL of dichloromethane was then added, and themixture was rotated overnight on a rotary evaporator apparatus at roomtemperature and without applied vacuum. The following morning, themixture was filtered on a sintered glass funnel, washed three times with30 mL of 20% methanol in dichloromethane, and dried under high vacuum.Following drying, 45 mL of a 35% solution of trifluoroacetic acid (TFA)in dichloromethane was added, and the resulting slurry was shaken atroom temperature for 45 minutes. The slurry was then filtered, and therecovered stationary phase was washed three times with 30 mL 20%methanol in dichloromethane, then with 30 mL of a solution of 10%Hunig's base in dichloromethane, then taken up in a slurry with2-propanol, and packed into a 4.6 mm id×25 cm length HPLC column forevaluation. Residual stationary phase taken from the column packerreservoir was dried overnight under high vacuum, then submitted forcombustion analysis (C 7.5%; N 2.0%).

EXAMPLE 6

In Situ Preparation of N-L-prolyl-3-aminopropyl silica

A commercial amino silica column (Chromegabond Amine; 5μ particle size;60 Å pore size; 4.6 mm column i.d.; 25 cm column length) was flushedwith dichloromethane at a flow rate of 2 mL/min for a period of 30minutes. A 1 M solution of the Boc-L-Pro in acetonitrile containing oneequivalent of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride was then passed through the column at a flow rate of 2mL/min for 50 minutes. The column was flushed with dichloromethane for10 minutes at 2 mL/min, followed by flushing with 20% methanol indichloromethane for 20 minutes at 2 mL/min. Removal of the Bocprotecting group was performed in situ by first flushing the column withdichloromethane at 5 mL/min for 10 minutes, followed by a solution of 4%trifluoroacetic acid in dichloromethane at 5 mL/min for 40 minutes,which is then flushed out with dichloromethane at 5 mL/min for 20minutes. Then, a solution of 0.5% triethylamine in dichloromethane ispumped through the column at 5 mL/min for 40 minutes, followed bydichloromethane at 5 mL/min for 20 minutes.

EXAMPLE 7 Comparison of HPLC of Pneumocandin B₀ on Different StationaryPhases at Low Loadings

Analytical scale HPLC columns, 250 mm long×4.6 mm id, were used tocompare the retention and selectivity characteristics obtained withdifferent stationary phases when using an analytical loading ofPneumocandin B₀ and its analogues. The different stationary phasesincluded: (1) regular silica, and (2) aminopropyl silica (obtained fromEka Chemicals (Bohus, Sweden)), and (3) Amide-80 (an amide moiety bondedto silica, obtained from Tosoh Biosep LLC. (Japan)). All these packingshad a 10 μm particle size and 120 Å pore diameter. The regular silicawas run as a simple liquid chromatography system, and using a mobilephase modified with L-proline, resulting in saturation of the silicawith proline. See J. Nti-Gyabaah, et al., “Large-scale purification ofpneumocandin B₀, a precursor for CANCIDAS”, PREP-2003, 16thInternational Symposium, Exhibit and Workshops on Preparative/ProcessChromatography, San Francisco, Calif., Wednesday, Jul. 2, 2003 or U.S.Provisional Application No. 60/422,356 filed Oct. 30, 2002.

The ternary mobile phase and the feed diluent (84/9/7 v/v/v ethylacetate, methanol and water) were made using HPLC grade solvents fromFisher Scientific (Pittsburg, Pa., USA). L-proline used for the prolineelution silica run was obtained from Ajinomoto (Japan). The L-prolinewas dissolved in the ternary mobile phase at 0.12 g/L. The feed wasprepared by blending pure Pn B₀ (crude Pn B₀ that had been purified bythe standard silica gel method) with aliquots of solutions containing PnB₅, Pn C₀ and Pn E₀, so that these analogs were each present at roughly10% the concentration of Pn B₀, which was present at roughly 1 g/L. ThePn C₀-enriched solution was obtained by selecting tailcut fractions froman injection of crude Pn B₀ on silica gel using the standard silica gelmethod. The Pn B₅- and Pn E₀-enriched solutions were obtained byselecting forecut fractions from an injection of Pn B₀ on silica gelusing the proline elution method, and then further purifying those cutsby a reversed-phase method similar to that employed for analyticalanalysis of Pn B₀ (described later in this example) but with higher feedloading. An Agilent HP-1100 HPLC system (Waldbronn, Germany) with diodearray detector was used for the HPLC runs, as well as for fractionanalysis; a wavelength of 278 nm was used for detection.

For each injection, 10 μL of the 1 g/L feed solution was injected. Themobile phase flow rate was 1.1 mL/min. For the run using theproline-modified mobile phase and regular silica, L-proline wasdissolved in the mobile phase at ˜0.12 g/L and ˜480 mL of the solutionwas then pumped through the silica column (to in situ coat the silicawith proline). The L-proline containing solution was used as eluent forthat run. Chromatograms obtained from the runs are shown in FIGS. 1, 2,3, and 4.

For each run, the peaks were identified by collecting fractions andanalyzing them by normal and reversed phase HPLC using methods describedin U.S. Provisional Application No. 60/422,356 filed Oct. 30, 2002(incorporated by reference) or J. Nti-Gyabaah, et al., “Large-scalepurification of pneumocandin B₀, a precursor for CANCIDAS”, PREP-2003,16th International Symposium, Exhibit and Workshops onPreparative/Process Chromatography, San Francisco, Calif., Wednesday,Jul. 2, 2003. The retention times of pneumocandins B₀, E₀, B₅ and C₀were then used to calculate the capacity factors, from which theselectivities between Pn B₀ and the three analogs were obtained. Theresults are shown in FIGS. 1-5 and Table 2. TABLE 2 Selectivitiesbetween Pneumocandin B₀ and analogs Selectivity (Relative to Pn B₀) PnE₀ Pn B₅ Pn C₀ FIGS. Bare silica 1.03 1.05 1.27 1 “Prolinated” silica*1.22 1.25 1.26 2 Aminopropyl-silica 1.67 1.66 1.06 3 Amide-80 silica1.46 1.42 1.25 4*silica gel chromatography using a proline-modified mobile phase

EXAMPLE 8 Purification of Pneumocandin B₀ Using Amide-80 Silica as theStationary Phase

Amide-80 bonded phase was obtained as a 250 mm long×4.6 mm id column (10am particle size, 8 nm pore size, spherical) from Tosoh Biosep LLC. Aseparation using the same feed material on a bare silica columnequilibrated with proline-modified mobile phase was performed as acontrol, using a W. R. Grace/Davison silica gel Grade-631 column (250 mmlong×4.6 mm id, 16-2(Tm particle size, 60 Å pore size, irregular)supplied by Princeton Chromatography. The mobile phase composition forboth runs was 88/917 v/v/v ethyl acetate/methanol/water (e/m/w); theproline-modified mobile phase also contained 0.12 g/L of L-proline. Allsolvents were HPLC grade from Fisher Scientific. Proline was obtainedfrom Ajinomoto (Japan).

A partially purified preparation of Pneumocandin B₀ (Pn B₀ crude) with apurity of 61.9% was used to prepare the column feed. The methods forpreparing Pn B₀ crude are given in U.S. Pat. Nos. 5,202,309, 5,194,377and 6,610,822. For the Amide-80 run, the feed solution was prepared bydissolving Pn B₀ crude into a 75/20/8 v/v/v mixture of ethyl acetate,methanol and water; the concentration of Pneumocandin B₀ in the feedsolution was ˜45 g/L. For the control run, a 75/17/8 v/v/v ethylacetate/methanol/water feed solvent mixture was employed, with 1.5 g/Lproline, and ˜45 g/L Pn B₀.

Before use, the columns were flushed with 10 column volume (cv) ofmethanol and equilibrated with 10 cv of the mobile phase. For eachinjection, 1.2 mL of the feed solution was injected (representing ˜15g/L bed). The mobile phase flow rate was ˜0.5 ml/min. For each run,fractions were collected and analyzed to assess the quality of theseparation. Each injection was repeated to ensure a consistent elutionprofile. For the run employing proline-modified mobile phase, ˜480 mL ofthe proline-containing mobile phase was pumped through the bare silicacolumn prior to the first injection.

Analytical Methods:

For each run, fractions were collected and analyzed by reversed andnormal phase HPLC. The samples were analyzed on an Agilent HP-1100analytical HPLC system. The reversed phase HPLC was used to quantifymost species, except for Pn C₀ which co-elutes with Pn B₀ in thereversed phase method. The normal phase HPLC assay was used to determinePn C₀.

Measurement of Pn C₀

The amount of Pn C₀ was determined by normal phase Pn B₀ assay, anisocratic HPLC method, which employs a YMC silica column(SL12S05-2546WT) with a particle size of 5 μm and a pore size of 120 Å.The column was 250×4.6 mm i.d. and maintained at 25° C. Elution wasisocratic with 84/9/7 ethyl acetate/methanol/water eluent. Flow rate was1.2 mL/min, and detection was by UV absorbance of 278 nm. Productsamples required no special preparation prior to injection.

Measurement of Pn B₀ and Other Species

The reversed phase HPLC assay is used to measure Pn B₀/Pn C₀ and theother species, including Pn E₀ and Pn B₅. The reversed phase assay usesa gradient HPLC method with an YMC J'Sphere column (JM08S04-2546WT),particle size of 4 μm and pore size of 80 Å. The column was 250×4.6 mmi.d. and maintained at 30° C. The two mobile phases used were 0.1%phosphoric acid (A) and acetonitrile (B). The elution gradient startedat 60% A and 40% B and ramped to 1% A and 99% B over 45 minutes at 1.5mL/min, with UV detection at 220 nm. Prior to analysis, samples wereblown dry under nitrogen and re-dissolved in methanol to the originalconcentration.

Results

A typical chromatogram obtained using the proline-modified mobile phaseon bare silica gel is shown in FIG. 6. The Pn B₀ elutes as a large richcut as indicated in the figure, with excellent resolution from Pn E₀ andPn B₅, which elute in the preceding peak.

A chromatogram obtained from a run on the Amide-80 column is shown inFIG. 7. No proline or other modifier was employed in either the mobilephase or feed solvent for this run. A slightly higher level of methanolis used in the feed solvent mixture than for the control case. Theretention of Pn B₀ on this solid phase was similar to that achieved forthe control run with the proline-modified mobile phase (shown in FIG.6). In fact, the elution profile appears very similar to the controlsystem, except that the main Pn B₀ peak is broader.

Many fractions were collected from ˜6 to 14 column volumes (cv) and thefractions were analyzed by both normal and reversed phase HPLC. Thisanalysis confirmed that analogues such as Pn E₀ and Pn B₅ are wellresolved from Pn B₀; these analogues were contained in fractionsrepresenting ˜6 to 8.5 cv in FIG. 7. Results of the normal phaseanalysis indicated that most of Pn C₀ was resolved into the fractionsrepresenting 13 to 14 cv in FIG. 7. Therefore, the fractionsrepresenting ˜8.5 to 13 cv, shown in FIG. 7, could be combined to give apure rich cut with yield >90%.

EXAMPLE 9 Purification of Pneumocandin B₀ Using N-L-prolyl-3-aminopropylsilica as the Stationary Phase

A column was prepared containing the N-L-prolyl-3-aminopropyl silicastationary phase; methods for preparing this stationary phase werepresented in Examples 5 and 6. The column utilized was 250 mm long×4.6mm id, and contained the proline-amide moiety bonded to spherical silicafrom ES Industries which had 5 μm particle size and 60 Å pore size. Theexperiment was otherwise identical to that described in Example 8, andthe results may also be evaluated against the control in that example asillustrated in FIG. 6.

The chromatogram obtained from the run on the N-L-prolyl-3-aminopropylsilica bonded phase is shown in FIG. 8. As illustrated, despite theincreased levels of methanol in the feed and the exclusion of prolinefrom the system, the retention of Pn B₀ on this phase was similar tothat achieved on bare silica using the proline-modified mobile phase(shown in FIG. 6). However, there are some key differences. First, thelarge solvent front peak normally observed in control system appearsdiminished. Another difference is that impurities that elute at theleading edge of the main Pn B₀ peak appear to be well separated fromeach other. Finally, there is more tailing of the Pn B₀ peak (comparedto FIG. 6).

Many fractions were collected from ˜10 to 21 cv and the fractions wereanalyzed by both normal and reversed phase HPLC. The analytical resultsindicated the key early eluting impurities such as Pn B₅ and Pn E₀ wereclearly resolved from Pn B₀. These impurities are mostly contained inthe fraction which represented the 10-13 cv range in FIG. 8. Results ofthe normal phase analysis indicate most of the Pn C₀ was resolved intothe fraction that represented the 13 to 14 cv range in FIG. 8.Therefore, ˜13 to 19 cv (shown in FIG. 8) could be combined to give apure rich cut with yield >90%.

EXAMPLE 10 Purification of Pneumocandin B₀ UsingN-methylcarbamoyl-3-aminopropyl Silica as the Stationary Phase

A column was prepared containing the N-methylcarbamoyl-3-aminopropylsilica stationary phase; methods for preparing this stationary phasewere presented in Examples 3 and 4. The column in this example was 250mm long×4.6 mm id, and contained the N-methylcarbamoyl-3-aminopropylsilica moiety bonded to spherical silica from ES Industries which had 5μm particle size and 60 Å pore size. The experiment was otherwiseidentical to that described in Example 8, and the results may also beevaluated against the control in that example as illustrated in FIG. 6.

A chromatogram obtained from the run on theN-methylcarbamoyl-3-aminopropyl silica bonded phase is illustrated inFIG. 9. Despite the increased methanol in the feed and the exclusion ofproline from the system, the retention of Pn B₀ on this phase wasexcellent (compared to FIG. 6). Many fractions were collected from ˜21to 40 cv. The fractions were analyzed by normal and reversed phase HPLC.The fraction analysis indicated that the early eluting analogues such asPn E₀ and Pn B₅ were well resolved from Pn B₀ and contained in thefractions representing ˜21 to 25 cv, shown in FIG. 9. Results of thenormal phase analysis indicated that the resolution of Pn B₀ from latereluting analogues such as Pn C₀ was comparable to that in the controlprocess. A ring-opened degradate, which was seen at only trace levels ifat all in the previous examples, was present at levels of a few percentin the rich cuts of this run. Other than this degradate, the laterfractions were very pure.

EXAMPLE 11 Purification of Pneumocandin B₀ Using N-β-alaninamidopropylSilica as the Stationary Phase

A column was prepared containing the N-β-alaninamidopropyl silicastationary phase; methods for preparing this stationary phase werepresented in Examples 1 and 2. The column used was 250 mm long×4.6 mmid, and contained one propylamide moiety bound to an aminopropyl moietybonded to spherical silica from ES Industries which had 5 μm particlesize and 60 Å pore size. The experiment was similar to that described inExample 8, but the stationary phase was significantly more retentivethan most of the others evaluated, and so a stronger mobile phase(75/17/8 ethyl acetate/methanol/water) was needed to completely elute PnB₀ from the column. The results may still be evaluated against thecontrol as illustrated in FIG. 6.

A chromatogram obtained from the run on the N-β-alaninamidopropyl silicabonded phase is shown in FIG. 10. It is important to point out that thelarge solvent front peak normally observed in the proline elution methodsystem appears diminished. Another difference is that the key impuritiesthat elute at the leading edge of the main Pn B₀ peak are well resolved.

Several fractions were collected from ˜6 to 13 cv. The fractions wereanalyzed by both normal and reversed phase HPLC. The analytical resultsindicated that the early eluting analogues such as Pn E₀ and Pn B₅ wereclearly resolved from Pn B₀. These impurities were mostly contained inthe fractions that represent ˜6 to 8 cv. Results of the normal phaseanalysis indicate that most of the Pn C₀ was resolved into the fractionsthat represent 11 to 13 cv. Fractions that represent ˜8 to 11 cv (shownin FIG. 10) could be combined to give a pure rich cut with >90% yield. Alow level of the ring-opened degradate was detected in the rich cutfractions. This is a potentially superior stationary phase, since itsretentivity allows use of a mobile phase for elution which is verysimilar in solvent strength to that of the feed solvent mixture, whichresults in very stable chromatographic performance.

1. A stationary phase of Formula I:

wherein R is: c) —(CH₂)_(n)CONH₂, or d) —COOR¹; n is: 1 to 4; and R¹ is:C₁-C₂ alkyl.
 2. The stationary phase of Formula I, as recited in claim1, selected from the group consisting of:


3. A method for the purification of a peptide or a lipopeptide by usinga liquid chromatography system with a stationary phase selected from thegroup consisting of: the stationary phase of Formula I, as recited inclaim 1, Tosoh Amide 80,

and a mobile phase, to improve the selectivity and/or productivity ofthe purification.
 4. The method as recited in claim 3, wherein themobile phase is a solvent system comprising one or more solvents.
 5. Themethod as recited in claim 4, wherein the solvents in the solvent systemare selected from the group consisting of: water, methanol, ethanol,isopropanol, hexane, heptane, ethyl acetate, isopropyl acetate,acetonitrile, methyl t-butyl ether (MTBE) and methylene chloride.
 6. Themethod as recited in claim 4, wherein the liquid chromatography systemis for the purification of a peptide.
 7. The method as recited in claim4, wherein the liquid chromatography system is for the purification of alipopeptide.
 8. The method as recited in claim 7, wherein thelipopeptide is a fermentation product precursor of caspofungin,micafungin, cilofungin, andulifungin and daptomycin.
 9. The method asrecited in claim 8, wherein the lipopeptide is the precusor ofcaspofungin, pneumocandin B₀.
 10. The method as recited in claim 9,wherein the mobile phase is a solvent system comprising water, methanol,and ethyl acetate.
 11. The method as recited in claim 6, wherein thepeptide is oxytocin or bradykinin.
 12. A method of purifyingPneumocandin B₀ with a liquid chromatography system comprising astationary phase selected from: Tosoh amide 80,

and a mobile phase to improve the selectivity and/or productivity of thepurification.
 13. The method as recited in claim 12, wherein the mobilephase comprises a solvent system, wherein the solvents in the solventsystem are selected from the group consisting of: water, methanol,ethanol, isopropanol, hexane, heptane, ethyl acetate, isopropyl acetate,acetonitrile, methyl t-butyl ether (MTBE) and methylene chloride. 14.The method as recited in claim 13, wherein the solvent system is amixture of ethyl acetate, methanol and water.